Transglutaminases and receptor tyrosine kinases

Transglutaminases are confounding enzymes which are known to play key roles in various cellular processes. In this paper, we aim to bring together several pieces of evidence from published research and literature that suggest a potentially vital role for transglutaminases in receptor tyrosine kinases (RTK) signalling. We cite literature that confirms and suggests the formation of integrin:RTK:transglutaminase complexes and explores the occurrence and functionality of these complexes in a large fraction of the RTK family

Structure-function studies of insulin-like growth factor I / by Gary Keith Shooter.

"20th of July 2001"Bibliography: leaves 124-165.xiv, 165 leaves : ill. (some col.), plates (col.) ; 30 cm.A study which aims at better understanding of the molecular mechanism involved in receptor binding, which may clarify the interdependence of IGF-I and insulin actions. The results suggest that factors other than receptor binding affinity may be important for determining the magnitude of the IGF-I mitogenic response. Insulin analogue studies demonstrate that the mitogenic response of the insulin receptor is related to the residence time of the ligand-receptor interaction rather than the receptor binding affinity. This study suggests that, like the insulin receptor, residence time rather than the equilibrium binding afinity may be an important factor in determining the magnitude of the mitogenic response from the type 1 IGF receptor.Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 200

A peptidomimetic inhibitor of matrix metalloproteinases containing a tetherable linker group

Successful wound repair and normal turnover of the extracellular matrix relies on a balance between matrix metalloproteinases (MMPs) and their natural inhibitors (the TIMPs). When over-expression of MMPs and abnormally high levels of activation or low expression of TIMPs are encountered, excessive degradation of connective tissue and the formation of chronic ulcers can occur. One strategy to rebalance MMPs and TIMPs is to use inhibitors. We have designed a synthetic pseudopeptide inhibitor with an amine linker group based on a known high-affinity peptidomimetic MMP inhibitor have demonstrated inhibition of MMP-1, -2, -3 and -9 activity in standard solutions. The inhibitor was also tethered to a polyethylene glycol hydrogel using a facile reaction between the linker unit on the inhibitor and the hydrogel precursors. After tethering, we observed inhibition of the MMPs although there was an increase in the IC50s which was attributed to poor diffusion of the MMPs into the hydrogels, reduced activity of the tethered inhibitor or incomplete incorporation of the inhibitor into the hydrogels. When the tethered inhibitors were tested against chronic wound fluid we observed significant inhibition in proteolytic activity suggesting our approach may prove useful in rebalancing MMPs within chronic wounds

A hydrogen tethered inhibitor of matrix metalloproteinases

During wound repair, the balance between matrix metalloproteinases (MMPs) and their natural inhibitors (the TIMPs) is crucial for the normal extra cellular matrix turnover. However, the over expression of several MMPs including MMP-1, 2, 3, 8, 9 and MMP-10, combined with abnormally high levels of activation or low expression of TIMPs, may contribute to excessive degradation of connective tissue and formation of chronic ulcers. There are many groups exploring strategies for promoting wound healing involving delivery of growth factors, cells, ECM components and small molecules. Our approach for improving the balance of MMPs is not to add anything more to the wound, but instead to neutralise the over-expressed MMPs using inhibitors tethered to a bandage-like hydrogel. Our in vitro experiments using designed synthetic pseudo peptide inhibitors have been demonstrated to inhibit MMP activity in standard solutions. These inhibitors have also been tethered to polyethylene glycol hydrogels using a facile reaction between the linker unit on the inhibitor and the gel. After tethering the inhibition of MMPs diminishes to some extent and we postulate that this arises due to poor diffusion of the MMPs into the gels. When the tethered inhibitors were tested against chronic wound fluid obtained against patients we observed over 40% inhibition in proteolytic activity suggesting our approach may prove useful in rebalancing MMPs within chronic wounds

Development of an enhanced proteomic method to detect prognostic and diagnostic markers of healing in chronic wound fluid

Background \ud \ud Chronic venous leg ulcers are a significant cause of pain, immobility and decreased quality of life for patients with these wounds. In view of this, research efforts are focused on multiple factors in the wound environment to obtain information regarding the healing of ulcers.\ud \ud Objectives \ud \ud Chronic wound fluid (CWF), containing a complex mixture of proteins, is an important modulator of the wound environment, and therefore we hypothesized that these proteins may be indicators of the status of wounds and their potential to heal or otherwise. To explore this we developed and validated a proteomic approach to analyse CWF.\ud \ud Methods \ud \ud In this study, pooled CWF was depleted of high abundant proteins using immunoaffinity chromatography. The flow-through and bound fractions were collected, concentrated, desalted and analysed using a range of techniques. Each fraction was further separated using two-dimensional (2D) gel electrophoresis and 2D liquid chromatography and analysed using mass spectrometry (MS).\ud \ud Results \ud \ud Western blot analysis against three high abundant proteins confirmed the selective removal of these proteins from CWF. Critically, one-dimensional and 2D gel electrophoresis indicated that subsequent removal of these proteins enhanced the ability to detect proteins in low abundance in CWF. Further, MS demonstrated that depletion of these abundant proteins increased the detection of other proteins in these samples.\ud \ud Conclusions \ud \ud Results obtained indicate that this approach significantly improves separation of proteins present in low concentrations in CWF. This will facilitate the identification of biomarkers in samples collected from patients with ulcers and lead to improved patient therapies and wound care approaches

Intensified downstream processing of monoclonal antibodies using membrane technology

The need to intensify downstream processing of monoclonal antibodies to complement the advances in upstream productivity has led to increased attention towards implementing membrane technologies. With the industry moving towards continuous operations and single use processes, membrane technologies show promise in fulfilling the industry needs due to their operational flexibility and ease of implementation. Recently, the applicability of membrane-based unit operations in integrating the downstream process has been explored. In this article, we review the major developments in the application of membrane-based technologies in the bioprocessing of monoclonal antibodies. We focus on the recent progress towards developing intensified end-to-end bioprocesses and the critical role membrane technology will play in achieving this goal. This article is protected by copyright. All rights reserved

Elevated uric acid correlates with wound severity

Chronic venous leg ulcers are a major health issue and represent an often overlooked area of biomedical research.\ud Nevertheless, it is becoming increasingly evident that new approaches to enhance healing outcomes may arise\ud through better understanding the processes involved in the formation of chronic wounds. We have for the first\ud time shown that the terminal purine catabolite uric acid (UA) is elevated in wound fluid (WF) from chronic venous\ud leg ulcers with relative concentrations correlating with wound chronicity. We have also shown a corresponding\ud depletion in UA precursors, including adenosine, with increased wound severity. Further, we have shown that\ud xanthine oxidase, the only enzyme in humans that catalyses the production of UA in conjunction with a burst of\ud free radicals, is active in chronic WF. Taken together, this provides compelling evidence that xanthine oxidase may\ud play a critical role in the formation of chronic wounds by prolonging the inflammatory process

Attenuation of protease activity in chronic wound fluid with bisphosphonate-functionalised hydrogels

Chronic ulcers are an important and costly medical issue, imposing considerable pain, reduced mobility and decreased quality of life. The common pathology in these chronic wounds is excessive proteolytic activity, resulting in degradation of key factors critical to the ulcer's ability to heal. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), with many authors suggesting that they need to be inhibited for the ulcer to heal. The studies we report here show that the excessive MMP activity in CWF can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and tethered to a poly(2-hydroxy methacrylate) (PHEMA) hydrogel. Furthermore, these functionalised alendronate hydrogels appear to be biologically inert as assessed in a three-dimensional ex vivo human skin equivalent model. Together, these results highlight the potential use of a tethered MMP inhibitor to inhibit protease activity in wound fluid. This approach may improve wound healing as it still allows MMPs to remain active in the upper cellular layers of the ulcer bed where they perform vital roles in wound healing; thus may offer an attractive new device-orientated wound therapy